The present invention relates to a microbiological method for the preparation of an epothilone.
Epothilones are macrolide compounds that find utility in the pharmaceutical field. For example, epothilones A and B having the structures: 
have been found to exert microtubule-stabilizing effects similar to paclitaxel (TAXOL(copyright)) and hence cytotoxic activity against rapidly proliferating cells, such as, tumor cells or other hyperproliferative cellular disease, see Bollag et al., Cancer Res., Vol. 55, No. 11, 2325-2333 (1995).
Epothilones A and B are natural anticancer agents produced by Sorangium cellulosum that were first isolated and characterized by Hofle et al., DE 4138042; WO 93/10121; Angew. Chem. Int. Ed. Engl. Vol. 35, No 13/14, 1567-1569 (1996); and J. Antibiot., Vol. 49, No. 6, 560-563 (1996). Subsequently, the total syntheses of epothilones A and B have been published by Balog et al., Angew. Chem. Int. Ed. Engl., Vol. 35, No. 23/24, 2801-2803, 1996; Meng et al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073-10092 (1997); Nicolaou et al., J. Am. Chem. Soc., Vol. 119, No. 34, 7974-7991 (1997); Schinzer et al., Angew. Chem. Int. Ed. Eng., Vol. 36, No. 5, 523-524 (1997); and Yang et al., Angew. Chem. Int. Ed. Engl., Vol. 36, No. 1/2, 166-168, 1997. PCT WO98/25929 disclosed the methods for chemical synthesis of epothilone A, epothilone B, analogs of epothilone and libraries of epothilone analogs. The structure and production from Sorangium cellulosum DSM 6773 of epothilones C, D, E, and F was disclosed in WO98/22461.
The present invention relate s to a method for obtaining epothilones with desired substituents at a terminal carbon position. In particular, the present invention provides a method for the preparation of hydroxyalkyl-bearing epothilones, which compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogs.
One embodiment of the invention provides a method for the preparation of at least one epothilone of the following formula I
HOxe2x80x94CH2xe2x80x94(A1)nxe2x80x94(Q)mxe2x80x94(A2)oxe2x80x94Exe2x80x83xe2x80x83(I)
where
A1 and A2 are independently selected from the group of optionally substituted C1-C3 alkyl and alkenyl;
Q is an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring;
n, m, and o are integers selected from the group consisting of zero and 1, where at least one of m or n or o is 1; and
E is an epothilone core;
comprising the steps of contacting at least one epothilone of the following formula II
CH3xe2x80x94(A1)nxe2x80x94(Q)mxe2x80x94(A2)oxe2x80x94Exe2x80x83xe2x80x83(II)
where A1, Q, A2, E, n, m, and o are defined as above;
with a microorganism, or an enzyme derived therefrom, which is capable of selectively catalyzing the hydroxylation of formula II, and effecting said hydroxylation.
In another embodiment, the present invention provides a method for the preparation of at least one epothilone of the following formula III: 
where
Q is selected from the group consisting of 
G1 is the following formula V
HOxe2x80x94CH2xe2x80x94(A1)nxe2x80x94(Q)mxe2x80x94(A2)oxe2x80x83xe2x80x83(V)
where
A1 and A2 are independently selected from the group of optionally substituted C1-C3 alkyl and alkenyl;
Q is an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring;
n, m, and o are integers independently selected from the group consisting of zero and 1, where at least one of m or n or o is 1;
W is O or NR6;
X is selected from the group consisting of O; H, OR7;
M is O, S, NR8, CR9R10;
B1 and B2 are selected from the group consisting of OR11, OCOR12;
R1-R5 and R12-R17 are selected from the group consisting of H, alkyl, substituted alkyl, aryl, and heterocyclo, and wherein when R1 and R2 are alkyl they can be joined to form a cycloalkyl;
R6 is selected from the group consisting of H, alkyl, and substituted alkyl;
R7 and R11 are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl, and dialkylarylsilyl;
R8 is selected from the group consisting of H, alkyl, substituted alkyl, R13Cxe2x95x90O, R14OCxe2x95x90O and R15SO2;
R9 and R10 are selected from the group consisting of H, halogen, alkyl, substituted alkyl, aryl, heterocyclo, hydroxy, R16Cxe2x95x90O, and R17OCxe2x95x90O;
the pharmaceutically acceptable salts thereof and any hydrates, solvates, or geometric, optical and stereoisomers thereof;
comprising the steps of contacting at least one epothilone of the following formula IV: 
where
Q, W, X, M, B1, B2, and R1-R17 are as defined above;
G2 is the following formula VI
CH3xe2x80x94(A1)nxe2x80x94(Q)mxe2x80x94(A2)oxe2x80x83xe2x80x83(VI)
where A1, Q, A2, n, m, and o are defined as above;
the pharmaceutically acceptable salts thereof and any hydrates, solvates, or geometric, optical and stereoisomers thereof;
with a microorganism or enzyme derived therefrom capable of selectively catalyzing the hydroxylation of G2 to G1, and effecting said hydroxylation.